IRF8 is expressed by GPs and MPs but not GMPs, and is not required for GP and MP production by GMPs. (A-B) IRF8 levels in GMPs (GMP-Ly6C⁻), total GMP-Ly6C⁺, GPs (GMP-Ly6C⁺CD115^lo), and MPs (GMP-Ly6C⁺CD115^hi), as well as neutrophils (CD11b⁺Ly6G^hi Ly6C^int) and monocytes (CD11b⁺Ly6G⁻Ly6C^hi) isolated from bone marrow, were assessed by western blotting using GAPDH as a loading control (A), and by intracellular flow cytometry (B). The mean fluorescence intensities (MFIs) were plotted for GMPs, IRF8⁺ GPs (GP⁺) and MPs (B, right panel). All data are representative of at least 3 independent experiments. (C-D) Neutrophils and monocytes in the bone marrow, spleen, and blood (C) and GMPs, GPs, and MPs in the bone marrow (D) of IRF8^−/− mice were assessed by flow cytometry. Bone marrow numbers are per leg (femur + tibia). Data are presented as mean plus standard deviation of 5 wild-type and 5 IRF8^−/− mice. Statistical significance was assessed by Student t test (**P < .01, ***P < .001). (E-G) One thousand GMPs per well were cultured in liquid media containing 20 ng/mL GM-CSF (E), 50 ng/ml G-CSF (F), or 50 ng/ml M-CSF (G), and cultures were analyzed by flow cytometry at 24 hours (F) or 48 hours (E, G) to monitor for the production of GPs and MPs (see supplemental Figure 8 for gating strategy). Two to 5 wells were pooled as necessary to permit flow cytometry analysis. Data are presented as mean plus standard deviation of at least 3 wild-type and 3 IRF8^−/− mice (GM-CSF and M-CSF cultures, 5 mice per group; G-CSF cultures, 3 mice per group). Statistical significance was assessed by Student t test (**P < .01, ***P < .001). n.s., not significant.